Method for Stimulating the Intestinal Flora

ABSTRACT

The present invention relates to methods for feeding and to compositions to be administered to infants delivered via caesarian section and in particular to the use of a) at least two different microorganisms; or b) at least one microorganism and at least one indigestible oligosaccharide; or c) at least two different Bifidobacteria species, subspecies or strains for the manufacture of a composition for enteral administration to an infant delivered via caesarean section. Thereby it is possible to stimulate the healthy development of the intestinal flora of said infants.

FIELD OF THE INVENTION

The present invention relates to methods for feeding and to compositionsto be administered to infants delivered via caesarian section.

BACKGROUND OF THE INVENTION

When formulating nutrition for infants, breast milk is used as the goldstandard. Ingredients in particular of non-human origin are normallycombined to mimic the compositional features and physiological effectsof the human breast milk.

One important aspect of human milk is that it provides energy and fluidsto the infant. Besides providing energy, human breast milk contains manyadditional components, which aim to maintain the health of the infant.Human breast milk for example contains prebiotic fiber, which stimulatesthe development of a healthy intestinal flora A healthy intestinal florahas numerous positive effects on the infant, such as a reduced incidenceof infections and a strengthened immune system.

Several commercially available infant milk formulae contain ingredientsthat aim to stimulate the development of the intestinal flora. Infantformulae can for example contain prebiotic fibers or live probioticorganisms. The prebiotic fibers normally pass undigested through theupper gastrointestinal tract and selectively stimulate the growth ofbeneficial bacteria in the colon. Live probiotic bacteria increasespecific bacterial counts in the intestine.

EP 1105002 describes a prebiotic carbohydrate mixture comprising one ormore soluble oligosaccharides and one or more soluble polysaccharides,with at least 80 wt. % of each being prebiotic.

WO 2005039319 describes a preparation comprising Bifidobacterium breveand a mixture of non-digestible carbohydrates for non- or partiallybreast-fed infants as well as the use thereof for the treatment orprevention of immune disorder in non- or partially breast-fed infants.

SUMMARY OF THE INVENTION

Infant formulae are normally designed to mimic the development of anintestinal flora in an infant receiving human breast milk, with theimplication that all infants react similar to human breast milk andinfant formula. However, the present inventors have found that asub-population of infants, namely those infants delivered via caesariansection, will react differently because their intestinal flora at birthis completely different from the intestinal flora of infants born viathe vaginal route. In particularly, the profile and content ofBifidobacteria species of infants delivered via caesarean section isdifferent from the intestinal profile and content of Bifidobacteriaspecies of infants delivered via the vaginal route.

The present inventors have analyzed the intestinal flora of newbornsafter caesarean delivery and newborns after vaginal delivery. It wassurprisingly found that great differences exist between the two groupsin composition of the intestinal flora. Particularly infants born viathe vaginal route contain at least about three different Bifidobacteriaspecies, whereas the infants born via cesarean section lack the mostimportant Bifidobacteria species.

It was also found that the infants born via caesarian sectionparticularly lack Bifidobacterium breve, Bifidobacterium infantis,Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacteriumadolescentis and Bifidobacterium longum. These species were present inthe flora of most infants born via the vaginal route.

Because the intestinal flora plays a crucial role in the development ofthe infant, in particularly the stimulation of the immune system andresistance against infections, it is of utmost importance to stimulatethe healthy development of the intestinal flora of infants born viacesarean section.

Furthermore the newborns delivered via caesarian section lacked abiodiversity in their intestinal flora. Only one or two differentspecies of Bifidobacteria were detected in infants born via c-section,while the intestinal flora of newborns delivered via the vaginal routenormally contains several different species of Bifidobacteria. Thepresent inventors believe that these observations are indicative for ageneral lack of a species variety in the intestinal tract of infantsdelivered via c-section. The biodiversity is of great importance for theachieving the desired physiological effects and optimally stimulate thehealth of the infant.

Natren® produces the probiotic product Life Start® which is designedspecifically for infants and suitable for infants delivered viacaesarean section. Life Start® is made with Bifidobacterium infantis.Because the Life Start® product contains only one single Bifidobacteriaspecies, the benefits for the infant will be very limited.

Particularly unexpected is also the observation that improvements ofinfant health can be achieved even when infants are fed with breastmilk. In the case where infants are delivered via cesarean section,breast milk is (in most cases) the best nutrition for the infant.However, the breast milk also does not instantaneously result in asimilar flora as obtained in infant born via vaginal delivery. Thepresent method is therefore also advantageously used when infant receivehuman breast milk.

Hence, the present invention particularly aims to: a) increase theoccurrence of particular species in the intestinal flora of infants bornvia caesarian section; b) increase the biodiversity of microorganisms inthe intestinal flora; and/or c) stimulate the growth of beneficialmicroorganisms, particularly Bifidobacteria.

Hence in one aspect the present invention provides a method forstimulating the healthy development of the intestinal flora of infantsborn via caesarian section by administrating a composition containing:

-   -   at least two different microorganisms;    -   at least one microorganism and at least one indigestible        oligosaccharide; or    -   at least two different Bifidobacteria species, subspecies or        strains.

The microorganism will increase the biodiversity of the infants flora,while the indigestible oligosaccharides stimulate the development andgrowth of inherent and administered microorganisms.

The composition to be administered in the present method preferablycontains multiple different bacterial species, preferably multipleBifidobacteria species. This results in the optimal stimulation of theintestinal flora of the caesarean delivered infants.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for feeding infants, said methodcomprising enterally administering a composition to an infant deliveredvia caesarean section, said composition comprising: a) at least twodifferent microorganisms; or b) at least one microorganism and at leastone indigestible oligosaccharide; or c) at least two differentBifidobacteria species, subspecies or strains.

In a further aspect the present invention provides a method forstimulating the health of an infant delivered via caesarean section,comprising administering to the infant and within 100 hours after birtha composition containing microorganisms and/or indigestibleoligosaccharides.

In still a further aspect the present invention provides a method forthe manufacture of infant nutrition suitable for infant born viacesarean section comprising admixing: A) human breast milk; and B) acomposition comprising: i) at least two different microorganisms; or ii)at least one microorganism and at least one indigestibleoligosaccharide; or iii) at least two different Bifidobacteria species,subspecies or strains.

In still a further aspect, the present invention provides a compositioncomprising at least four Bifidobacteria species, subspecies or and/orstrains selected from the group consisting of Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacteriumcatenulatum, Bifidobacterium adolescentis, Bifidobacterium thermophilum,Bifidobacterium gallicum, Bifidobacterium animalis, Bifidobacteriumangulatum, Bifidobacterium pseudocatenulatum, Bifidobacteriumthermacidophilum and Bifidobacterium longum.

The present invention also provides a nutritional composition comprisingbetween 5 and 25 en % protein; between 25 and 60 en % fat; between 30and 70 en % carbohydrate; at least two different species ofBifidobacteria and at least one species of Lactobacillus.

The present invention also provides a container with a liquid volumebetween 0.5 and 50 ml containing a composition to be administered to aninfant comprising at least two different microorganisms; or at least onemicroorganism and at least one indigestible oligosaccharide; or at leasttwo different Bifidobacteria species, subspecies or strains.

In a further aspect the present invention provides a method forstimulating the development of a healthy intestinal flora in an infantcomprising the step of: A) admixing I) a nutritionally orpharmaceutically acceptable liquid; and II) a dry composition, whereinthe dry composition II comprises at least two different microorganisms;or at least one microorganism and at least one indigestibleoligosaccharide; or at least two different Bifidobacteria species,subspecies or strains; and B) administering the composition obtained instep a) to an infant born via caesarean section.

The composition used in the present invention are preferably nutritionaland/or pharmaceutical compositions and suitable for administration toinfants.

Caesarian Section

The present invention relates to the enteral administration of acomposition containing a microorganism to infants delivered viacaesarean section. A caesarean section (c-section) is a surgicalprocedure where an infant is delivered through an incision made in themother's abdominal wall, and then through the wall of the uterus. Acaesarean section is usually performed when it is safer for the motheror the infant than a vaginal delivery. Other times, a woman may chooseto have a caesarean section rather than deliver her infant vaginally.

Diversity

The present composition contains at least two different microorganisms;or at least one microorganism organism and at least one indigestibleoligosaccharide; or at least two different Bifidobacteria species,subspecies or strains. The microorganisms are preferably bacteria and/oryeasts. Preferably the microorganisms used in the present invention areprobiotic, i.e. when applied to man or animal, it beneficially affectsthe host by improving the properties of the indigenous microflora. Theabove-mentioned combinations commonly aim to increase the diversityand/or the quantity of microorganisms in the intestine of the cesareansection delivered infant. This beneficially affects the infant, provingnumerous health benefits.

The term “different microorganisms” as used for the present inventionrefers to microorganism belonging to a different genus and/or species.Preferably the different microorganisms are different bacteria.Preferably, the different microorganisms are different species. Forexample and preferably, the present composition comprises or consists ofBifidobacterium breve (B. breve) and Bifidobacterium catenulatum (B.catenulatum). B. breve and B. catenulatum are herein considered as twodifferent bacterial species. Bacterial species are not considereddifferent if these are different subspecies. For example, Lactobacillusdelbrueckii subspecies delbrueckii and Lactobacillus delbrueckiisubspecies bulgaricus are not considered two different species. Thepresent composition contains at least two different microorganisms,preferably at least two different bacterial species. Preferably thepresent composition contains at least three different species, morepreferably at least four different species. Preferably the microorganismor bacterial species used are probiotic.

In one embodiment the present composition contains at least twodifferent Bifidobacteria species, subspecies or strains. The presentcomposition preferably comprises at least one, more preferably at leasttwo, even more preferably at least three, most preferably at least fourdifferent Bifidobacterium strains. The present composition preferablycomprises at least one, more preferably at least two, even morepreferably at least three, most preferably at least four differentBifidobacteria subspecies. For example Bifidobacterium animalissubspecies animalis and Bifidobactrium animalis subspecies lactis aredifferent subspecies. B. breve M16V en B. breve R0070 are differentstrains.

Microorganisms

Preferably the present composition contains at least one microorganismsselected from the group consisting of lactic acid bacteria, bacilli andyeasts, preferably at least one lactic acid bacteria selected from thegroup consisting of Carnobacterium, Enterococcus, Lactobacillus,Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus,Tetragenococcus, Vagococcus and Weissella and Bifidobacteria. Thepresent composition preferably contains at least two, more preferably atleast three, most preferably at least four different microorganisms.

Preferably the present composition contains at least one speciesselected from the genus Bifidobacteria, more preferably at least two,even more preferably at least three, more preferably at least four, mostpreferably at least five species from the genus of Bifidobacteria.Bifidobacteria are Gram-positive, anaerobic, rod-shaped bacteria Thepresent Bifidobacterium species preferably have at least 95% identity ofthe 16 S rRNA sequence when compared to the type strain of therespective Bifidobacterium species, more preferably at least 97%identity as defined in handbooks on this subject for instance Sambrook,J., Fritsch, E. F., and Maliatis, T. (1989), Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory Press.The Bifodobacteria preferably used are also described by Scardovi, V.Genus Bifidobacterium. p. 1418-p. 1434. In: Bergey's manual ofsystematic Bacteriology. Vol. 2. Sneath, P. H. A., N. S. Mair, M. E.Sharpe and J. G. Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635 p.

The present composition preferably comprises at least one, morepreferably at least two, even more preferably at least three, mostpreferably at least four Bifidobacterium species preferably selectedfrom the group consisting of Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium bifidum, Bifidobacterium catenulatum,Bifidobacterium adolescentis, Bifidobacterium thermophilum,Bifidobacterium gallicum, Bifidobacterium animalis, Bifidobacteriumangulatum, Bifidobacterium pseudocatenulatum, Bifidobacteriumthermacidophilum and Bifidobacterium longum. More preferably the presentcomposition preferably comprises at least one, more preferably at leasttwo, even more preferably at least three, most preferably at least fourBifidobacterium species preferably selected from the group consisting ofBifidobacterium breve, Bifidobacterium infantis, Bifidobacteriumbifidum, Bifidobacterium catenulatum, Bifidobacterium adolescentis andBifidobacterium longum. Most preferably the present composition containsBifidobacterium breve and/or Bifidobacterium catenulatum.

In a further preferred embodiment, the present composition comprises atleast one, more preferably at least two, even more preferably at leastthree, most preferably at least four Bifidobacterium subspecies. Instill a further preferred embodiment, the present composition comprisesat least one, more preferably at least two, even more preferably atleast three, most preferably at least four Bifidobacterium strains.

In a further preferred embodiment, the present composition contains alactic acid bacteria, preferably at least a bacteria selected from thegroup consisting of Lactobacilli, Lactococci and Streptococci. Morepreferably the present composition contains at least one bacteriaselected from the group consisting of Lactobacillus plantarum,Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus casei,Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillusfermentum, Lactobacillus lactis, Streptococcus thermophilus andLactobacillus paracasei. The further increased biodiversity will have astimulatory effect on health of the newborn.

The present composition preferably contains between 10¹ and 10¹³ colonyforming units (cfu) microorganisms per gram dry weight of the presentcomposition, preferably between 10² and 10¹², more preferably between10³ and 10¹⁰. Preferably, the present composition contains between 10¹and 10¹³ colony forming units (cfu) Bifidobacteria per g dry weight ofthe present composition, more preferably between 10² and 10¹², mostpreferably between 10³ and 10¹².

The present method preferably comprises the administration of a servingcontaining between 10¹ and 10¹³ cfu microorganisms more preferablybetween 10² and 10¹¹, most preferably between 10³ and 10¹⁰. The presentmethod preferably comprises the administration of a serving containingbetween 10¹ and 10¹³ cfu Bifidobacteria, more preferably between 10² and10¹², most preferably between 10³ and 10¹¹.

Oligosaccharides

The term “indigestible oligosaccharides” as used in the presentinvention refers to oligosaccharides/carbohydrates which are not or onlypartially digested in the intestine by the action of acids or digestiveenzymes present in the human upper digestive tract (small intestine andstomach) but which are fermented by the human intestinal flora.Preferably the present indigestible oligosaccharide has a degree ofpolymerisation (DP) of 2 to 100, preferably a DP 2 to 50.

Preferably the present indigestible oligosaccharide is a prebioticfiber. The term “prebiotic fiber” refers to non-digestible fibers thatbeneficially affects the host by selectively stimulating the growthand/or activity of one or a limited number of bacterial species in thecolon.

Preferably the present indigestible oligosaccharide is soluble. The term“soluble” as used herein, when having reference to a polysaccharide,fibre or oligosaccharide, means that the substance is at least 50%soluble according to the method described by L. Prosky et al., J. Assoc.Off. Anal. Chem. 71, 1017-1023 (1988).

Preferably the present composition contains at least one oligosaccharideselected from the group consisting of galactooligosaccharides,indigestible dextrins, xylooligosaccharides, arabinooligosaccharides,glucooligosaccharides, mannooligo-saccharides, isomalto-oligosaccharideand fructopolysaccharide.

The term “fructopolysaccharide” as used herein refers to apolysaccharide carbohydrate comprising a chain of at least 3 β-linkedfructose units, with a DP between 3 and 300, preferably between 20 and150. Preferably inulin is used. Inulin is available under the tradename“Raftilin HP®”, (Orafti). The average DP of the fructopolysaccharide ispreferably at least 15, more preferably at least 20 or more, up to 300.In inulin the fructose units are linked with a β(2→1) linkage.

Indigestible polydextins refer to digestion-resistant (malto)dextrins ordigestion-resistant polydextrose which have a DP of 10 to 50, preferablybetween 10 and 20. The indigestible polydextrins comprise α(1→4), α(1→6)glucosidic bonds and 1→2 and 1→3 linkages Indigestible polydextrins arefor example available under the tradename “Fibersol 2®” from MatsutamiInductries or Litesse® from Danisco.

The present inventors found that galactooligosaccharides can beadvantageously used in the present composition, because theseoligosaccharides where particularly effective in stimulating the growthof Bifidobacteria. Hence, in a preferred embodiment the presentcomposition contains galactooligosaccharides. The term“galactooligosaccharide” as used herein refers to an indigestiblesaccharide, wherein at least 30% of the saccharide units are galactoseunits, preferably at least 50%, more preferably at least 60%. Preferablythe saccharides of the galactooligosaccharide are β-linked, as is thecase in human milk.

Preferably the present composition contains a galactooligosaccharideselected from the group consisting of transgalactooligosaccharides,lacto-N-tetraose (LNT) and lacto-N-neotetraose (neo-LNT). In aparticularly preferred embodiment the present method comprises theadministration of transgalactooligosaccharide ([galactose]_(n)-glucose;wherein n is an integer between 1 and 60, i.e. 2, 3, 4, 5, 6, . . . ,59, 60; preferably n is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10).Transgalactooligosaccharides (TOS) are for example sold under thetrademark Vivinal™ (Borculo Domo Ingredients, Netherlands). Preferablythe saccharides of the transgalactooligosaccharides are β-linked.

The present composition preferably contains 0.5 to 75 grams of theindigestible soluble oligosaccharides per 100 gram dry weight,preferably between 0.5 and 50 grams. The present composition preferablycomprises 0.1 to 95 grams of the galactooligosaccharides per 100 gramdry weight, preferably between 0.1 and 50 grams.

The present method preferably comprises the administration of a servingcontaining between 0.05 and 25 grams indigestible oligosaccharide,preferably between 0.1 and 5 grams. The present method preferablycomprises the administration of a serving containing between 0.05 and 25grams galactooligosaccharides, preferably between 0.1 and 5 gramgalactooligosaccharides.

The present inventors have also found that a mixture of a long chainindigestible oligosaccharides and short chain indigestibleoligosaccharides synergistically stimulate the growth of a healthyintestinal flora, particularly Bifidobacteria. Hence the presentcomposition preferably contains at least two oligosaccharides withdifferent average degrees of polymerization (DP). Preferably the weightratios:

-   a. (indigestible oligosaccharides with DP 2 to 5): (indigestible    oligosaccharides with DP 6, 7, 8, and/or 9)>1; and-   b. (indigestible oligosaccharides with DP 10 to 60): (indigestible    oligosaccharides with DP 6, 7, 8, and/or 9)>1

Preferably both weight ratios are above 2, even more preferably above 5.

For further improvement, the oligosaccharide preferably has a relativelyhigh content of short chain oligosaccharides, as these stronglystimulate the growth of Bifodobacteria. Hence, preferably at least 10wt. % of the oligosaccharides in the present composition has a DP of 2to 5 (i.e. 2, 3, 4, and/or 5) and at least 5 wt. % has a DP of 10 to 60.Preferably at least 50 wt. %, more preferably at least 75 wt. % of theoligosaccharides have a DP of 2 to 9 (i.e. 2, 3, 4, 5, 6, 7, 8, and/or9).

To improve the biodiversity and stimulate the growth of multipleintestinal organisms, the present composition preferably comprises twooligosaccharides with a different structure. The present compositioncomprises at least two different oligosaccharides, wherein theoligosaccharides have a homology in saccharide units below about 90%,preferably below 50%, even more preferably below 25%, even morepreferably below 5%. The term “homology” as used in the presentinvention is the cumulative of the percentage of same saccharide unit inthe different oligosaccharides. For example, oligosaccharide 1 (OL1) hasthe structure fruc-fruct-glu-gal, and thus comprises 50% fruc, 25% galand 25% glu. Oligosaccharide 2 (OL2) has the structure fruc-fruc-glu,and thus comprises 66% fruc, 33% glu. The different oligosaccharidesthus have a homology of 75% (50% fruc+25% glu).

Preferably the present composition contains galactooligosaccharides andfructopolysaccharides.

Application

The present composition is preferably enterally administered, morepreferably orally. The present composition is therefore preferably aliquid. The term “enteral” here used also encompasses a rectal or analadministration.

When infants receive infant milk formula, the present composition withmicroorganisms and optionally including at least one indigestibleoligosaccharide, is preferably included in the nutritional formula. Theprobiotic and/or prebiotic may be separately added to the infantformula. When newborns receive nutrition via a tube, the microorganismscan be suitably included in the nutrition administered via tube.Stimulating diversity is of great importance, thus the present inventionalso provides a nutritional composition comprising between 5 and 25 en %protein; between 25 and 60 en % fat; between 30 and 70 en %carbohydrate; at least two different species of Bifidobacteria and atleast one species of Lactobacillus.

When infants receive human breast milk, the present composition can besuitably admixed with human breast milk The present inventionconsequently also provides a method for the manufacture of infantnutrition suitable for infant born via cesarean section comprisingadmixing: A) human breast milk; and B) a composition comprising: (i) atleast two different microorganisms; or (ii) at least one microorganismand at least one indigestible oligosaccharide; or (iii) at least twodifferent Bifidobacteria species, subspecies or strains. The preferredcharacteristics of composition B) are as provided hereinabove.

In a further preferred embodiment the present composition isadministered to the infant in a very small volume, e.g. by “inoculating”the infant. Preferably, the present composition is administered to theinfant with a syringe, pipette or tube, preferably directly after birth.In a further preferred embodiment, the present composition is rectallyor anally administered to the infant delivered via caesarean section,preferably in the form of a suppository, pill or tablet. Hence, thepresent invention also provides a suppository, pill or table suitablefor rectal administration to a infant with the age below one year,wherein said suppository, pill or tablet contains microorganisms and/orindigestible oligosaccharides, preferably the present composition asdescribed hereinabove.

In a further preferred embodiment the present invention provides amethod stimulating the intestinal flora of an infant comprising rectallyadministering to an infant with the age below 3 year a compositioncomprising microorganism and/or indigestible oligosaccharide. Preferablythe infant has an age below 1 year, more preferably below 2 weeks.Preferably the infant is delivered via caesarean section. Preferablythis composition for rectal administration contains the above-describedcomposition.

This procedure has the advantage that it does not interfere with thenormal breast-feeding practice and has high resemblance with vaginalinoculation, which occurs during birth. The present invention alsoprovides a method for stimulating the health of an infant delivered viacaesarean section, comprising administering to the infant and within 100hours after birth, preferably within 72 hours after birth, mostpreferably within 48 hours after birth, a composition containingmicroorganisms and/or indigestible oligosaccharides.

The composition is preferably suitable for administration directly afterbirth. Hence, in a further preferred embodiment, the present inventionprovides a container comprising a liquid composition with a volumebetween 0.5 and 50 ml, which contains the present composition, Theliquid with the present microorganism, optionally combined withindigestible oligosaccharides, can be suitably used in the presentmethod. Preferably the liquid has a volume between 0.5 and 25 ml. Thisvolume is preferably small, because it otherwise could interfere withthe appetite and drinking behaviors of the infant.

Similarly and encompassed by this invention is a container with areconstitutable dry composition containing the present composition,wherein the container has a volume of between 0.5 and 50 ml. Thiscontainer is preferably accompanied with instruction to reconstitute thepowder in a small volume of liquid, e.g. water.

The present invention thus also provides a method for stimulating thedevelopment of a healthy intestinal flora in an infant comprising stepA: admixing I) an in particular nutritionally or pharmaceuticallyacceptable liquid; and II) a dry composition, wherein the drycomposition II comprises at least two different microorganisms; or atleast one microorganism and at least one indigestible oligosaccharide;or at least two different Bifidobacteria species, subspecies or strains;and step B) administering the composition obtained in step a) to aninfant born via caesarean section.

The present composition is preferably administered to the infant in thefirst year of life, preferably within two weeks after birth, even morepreferably within one week after birth, most preferably within 48 hoursafter birth.

EXAMPLES Example 1 Molecular Characterization of Intestinal Microbiotain Infants born By Vaginal Delivery vs. Caesarean Delivery

In the present study the influence of mode of delivery (caesareandelivery versus vaginal delivery) on the intestinal microbialcomposition at the third day of life by using PCR-Denaturing GradientGel Electrophoresis (DGGE) and PCR-Temperature Gradient GelElectrophoresis (TGGE). Both DGGE and TGGE analyses have been utilized,together with the specific amplifications for ten Bifidobacteriumspecies.

After written informed consent had been obtained from the parentstwenty-three newborns after caesarean delivery and vaginal delivery havebeen enrolled in the study. The faecal samples were obtained on thethird day of life.

The microbial DNA was extracted and analysed according to Favier et al,Environ Microbiol 2002; 68:219-226 and Satokari et al, Appl EnvironMicrobiol 2001; 67:504-513; Satorkari et al System Appl Microbiol 2003;26:572-584.

The results of the Bifidobacteria detected in faecal samples of newbornsafter caesarean delivery obtained at the 3rd day of life are given inTable 1. Table 2 gives the Bifidobacteria detected in faecal samples ofnewborn after vaginal delivery obtained at the 3rd day of life.

It can be seen that the microbial flora of a infant born via caesareansection shows strong differences with a infant born via the vaginalroute. Recognition of these differences on species level enabled thepresent inventors to design the present compositions and methods.

These results are indicative for the advantageous use of the compositionand method according to the present invention, e.g. a method for feedingbabies born via caesarean section, stimulating a healthy intestinalflora and consequently preventing infection and stimulating a healthyimmune system.

TABLE 1 Caesarean B. B. B. B. B. B. B. catenulatum B. B. B. NEWBORNbreve infantis dentium angulatum bifidum lactis group adolescentislongum gallicum 1 − − − − − − − − − − 2 − − − − − − − − ++ − 3 − − − − −− − − − − 4 − − − − − − − − − − 5 − − − − − − − − − − 6 − − − − − − − −− − 7 − − − − − − − − − − 8 − − − − − − − − − − 9 − − − − − − − − − − 10− − − − − − − − − ++ 11 − − − − − − − − − − 12 − − − − − − − − − − 13 −− − − − − − − − − 16 − − − − − − − − − − 17 − − − − − − − − − − 18 − − −− − − − − − − 19 − − − − − − − − − − 20 − − − − − − − − − − 21 − − − − −− − − − − 22 − − − − − − − − − − 23 − − − − − − − − − − (−) = noamplification (+/−) = weak amplification (+) = positive amplification(++) = strong amplification

TABLE 2 Normal B. B. B. B. B. B. B. catenulatum B. B. B. NEWBORN breveinfantis dentium angulatum bifidum lactis group adolescentis longumgallicum  1a − + − − − − − − ++ −  2a +/− − − − ++ − ++ − ++ −  3a − − −− − − + − − −  4a +/− − − − − − ++ + + −  5a +/− − − − ++ − ++ − ++ ++ 6a − − − − +/− − ++ − ++ −  7a − − − − +/− − ++ ++ − −  8a ++ ++ − − −− + ++ − −  9a − − − − − − + ++ + − 10a ++ − − − − − + + − − 11a ++ − −− ++ − ++ − ++ − 12a + + − − + − + − ++ − 13a +/− − − − − − + − + − 16a− − − − − − ++ − + − 17a +/− − − − + − + − + − 18a +/− − − − + − + − + −19a + − − − − − + − + − 20a − − − − − − + − + − 21a − − − − − − + ++ + −22a − + − − − − ++ − + − 23a + − − − ++ − ++ − + − (−) = noamplification (+/−) = weak amplification (+) = positive amplification(++) = strong amplification

Example 2 In Vitro Fermentation of Galacto-Oligosaccharide by Infant'sFaeces

Aim: The capacity of galactooligosaccharides (GOS) and a combination ofGOS and inulin to favor the activity of lactic acid bacteria wasevaluated using an in vitro semi dynamic batch fermentation system usinginfants faeces. The amount of lactate was determined, since this is thefermentation product of lactic acid bacteria including Bifidobacteria.

Method: Fresh faeces was obtained from healthy bottle-fed babies. Freshfaecal material from babies ranging 1 to 4 months of age was pooled andput into a preservative medium (buffered peptone 20.0 g/l,L-Cysteine-HCl 0.5 g/l, Sodium thioglycollate 0.5 g/l, resazurine tablet1/1, pH 6.7) within 1 h and stored at 4° C. for at most 2 h before thefermentation experiment was started.

The preserved solution of faeces was centrifuged at 13,000 rpm for 15min. The supernatant was removed and the faces was mixed with McBain &MacFarlane medium (Buffered peptone water 3.0 g/l, yeast extract 2.5g/l, mucin (brush borders) 0.8 g/l, Tryptone 3.0 g/l, L-Cysteine-HCl 0.4g/l, bile salts 0.05 g/l, K₂HPO₄.3H₂O 2.6 g/l, NaHCO₃ 0.2 g/l, NaCl 4.5g/l, MgSO₄.7H₂O 0.5 g/l, CaCl₂ 0.228 μl, FeSO₄ 0.005 g/l), which isrepresentative for the intestinal environment, in a weight ratio of 1:5.

At t=0 15 ml of the faecal suspension was combined with 500 mg prebioticin a bottle and mixed thoroughly. As a control no prebiotic was added.The prebiotics added were as followed:

Media: 1 2 3 GOS: 0 500 450 Inulin: 0 0 50

As a source of GOS, Vivinal GOS (Borculo Domo) was used. As a source ofinulin RaftilinHP (Orafti) was used.

15 ml was transferred into a dialysis tube in a 250 ml bottle filledwith 250 ml of buffered medium (K₂HPO₄.3H₂O 2.6 g/l, NaHCO₃ 0.2 g/l,NaCl 4.5 g/l, MgSO₄.7H₂O 0.5 μl, CaCl₂ 0.228 g/l, FeSO₄.7H₂O 0.005 g/l,pH 6.3(?)). The bottle was closed and incubated at 37° C. Samples of 1ml were taken from the dialysis tube and from the dialysis buffer with ahypodermic syringe after 3 h and stored at −18° C. Experiments wereperformed in duplo.

Lactate was determined enzymatically, using a L-lactate acid detectionkit with D- and L-lactate-dehydrogenase (Boehringer Mannheim, Mannheim,Germany).

Results and Conclusions: The results are expressed as amounts of lactateformed per g of prebiotic added. The results show that the amount oflactate is increased when an infant's microflora ferments GOS or amixture of GOS and inulin. When no prebiotic was added, lactateproduction is not observed. With GOS alone 0.1801 mmol/g prebioticlactate is produced. When a combination of GOS and inulin is added thelactate produced is 0.2119 mmol per g prebiotic.

The results indicate that the oligosaccharides stimulate the growthand/or activity of lactic acid bacteria in infants, includingBifidobacteria. These results are indicative for the advantageous use ofthe indigestible oligosaccharides, particularly galactooligosaccharides,in the present composition and method according to the presentinvention, i.e. in a method for feeding babies born via caesareansection.

Example 3 Method for Feeding Babies Born Via Caesarean Section

Within two days after the infant is born via caesarean section, anutritional composition is administered which contains per 100 ml readyto feed formula: 1.6 gram protein, 3.6 gram fat, 6.4 gram digestiblecarbohydrates (mainly lactose), 0.8 gram non-digestible carbohydrates ofwhich 0.60 gram transgalactooligosaccharides, 0.07 gram inulin, 1×10⁹cfu Bifidobacterium breve, 1×10⁹ cfu Bifidobacterium catenulatum, 1×10⁹cfu Bifidobacterium longum and 1×10⁹ cfu Lactobacillus paracasei.

Example 4 Packaged Composition

Packaged composition wherein the package indicates that the product isparticular suitable for oral feeding infants born via Caesarean section,said composition comprising Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium bifidum, Bifidobacterium catenulatum,Bifidobacterium adolescentis, Bifidobacterium longum,galactooligosaccharides and inulin.

Example 5 Packaged Composition

Composition as in Example 4, wherein the composition has a volume of 10ml and is packed in a syringe.

Example 6 Rectal Administration

Pll suitable for rectal administration to an infant within two weeksafter birth, comprising galactooligosaccharides and Bifidobacteriumbreve, Bifidobacterium infantis, Bifidobacterium bifidum.

1-16. (canceled)
 17. A method for feeding infants which comprisesenterally administering to an infant a composition comprising: a) atleast two different microorganisms; or b) at least one microorganism andat least one indigestible oligosaccharide; or c) at least two differentBifidobacteria species, subspecies or strains for the manufacture of acomposition for enteral administration to an infant delivered viacaesarean section.
 18. The method according to claim 17, wherein thecomposition contains at least one species of Bifidobacteria.
 19. Themethod according to claim 17, wherein the composition comprises at leastone Bifidobacterium selected from the group consisting ofBifidobacterium breve, Bifidobacterium infantis, Bifidobacteriumbifidum, Bifidobacterium catenulatum, Bifidobacterium adolescentisBifidobacterium thermophilum, Bifidobacterium gallicum, Bifidobacteriumanimalis, Bifidobacterium angulatum, Bifidobacterium pseudocatenulatum,Bifidobacterium thermacidophilum and Bifidobacterium longum.
 20. Themethod according to claim 19, wherein the composition comprises at leasttwo Bifidobacterium species selected from the group as mentioned inclaim
 19. 21. The method according to claim 17, wherein the compositioncomprises at least one microorganism and at least one indigestibleoligosaccharide.
 22. The method according to claim 17, wherein thecomposition comprises an indigestible oligosaccharide selected from thegroup consisting of galactooligosaccharides, indigestible dextrins,xylooligosaccharides, arabinooligosaccharides, glucooligosaccharides,mannooligo-saccharides, isomalto-oligosaccharide andfructopolysaccharides.
 23. The method according to claim 17, wherein thecomposition comprises between 5 and 25 energy % protein, between 25 and60 energy % fat and between 30 and 70 energy % carbohydrate.
 24. Themethod according to claim 17, wherein the composition is administered toan infant receiving human breast milk.
 25. The method according to claim17, wherein the composition is administered to the infant within oneweek after birth.
 26. Nutritional composition comprising between 5 and25 energy % protein; between 25 and 60 energy % fat; between 30 and 70energy % carbohydrate; at least two different species of Bifidobacteriaand at least one species of Lactobacillus.
 27. The method of claim 17wherein the infant was born via cesarean section.
 28. The method ofclaim 17 wherein the composition is obtained by a process comprising thesteps of: a) admixing 1) a nutritionally or pharmaceutically acceptableliquid; and II) a dry composition, wherein the dry composition 11comprises at least two different microorganisms; or at least onemicroorganism and at least one indigestible oligosaccharide; or at leasttwo different Bifidobacteria species, subspecies or strains.
 29. Themethod of claim 17 wherein the composition containing microorganisms andindigestible oligosaccharides for the manufacture of a composition forstimulating the intestinal flora is of an infant delivered via caesareansection, by administering said composition to the infant and within 100hours after birth.
 30. The method of claim 17 wherein the compositioncomprising microorganisms and/or indigestible oligosaccharides for themanufacture of a composition for stimulating the intestinal flora of aninfant delivered via caesarean section by rectally administering saidcomposition to an infant with the age below 3 year.
 31. Nutritional orpharmaceutical composition comprising at least five species from thegenus of Bifidobacteria wherein at least four Bifidobacteria species,subspecies or and/or strains are selected from the group consisting ofBifidobacterium breve, Bifidobacterium infantis, Bifidobacteriumbifidum, Bifidobacterium catenulatum, Bifidobacterium adolescentis,Bifidobacterium thermophilum, Bifidobacterium gallicum, Bifidobacteriumanimalis, Bifidobacterium angulatum, Bifidobacterium pseudocatenulatum,Bifidobacterium thermacidophilum and Bifidobacterium longum. 32.Container with a liquid volume between 0.5 and 25 ml containing acomposition to be administered to an infant comprising at least twodifferent microorganisms; or at least one microorganism and at least oneindigestible oligosaccharide; or at least two different Bifidobacteriaspecies, subspecies or strains wherein the composition comprises atleast one species selected from the genus Bifidobacteria.